6243631Broad Institute2084-01_Inhibitor_Dose_DryPowder_Activity_Set5201372MITF Measured in Cell-Based System Using Plate Reader - 2084-01_Inhibitor_Dose_DryPowder_Activity_Set5Keywords: MITF, TRPM-1, melanoma, SK-MEL-5, luciferase reporterAssay Overview:The primary HTS will be performed using SK-MEL-5 melanoma cells stably expressing a construct that contains the TRPM1 promoter upstream of a luciferase reporter gene. TRPM-1 is a known target of the transcription factor, microphtalmia-associated transcription factor (MITF). The assay in its current format utilizes plating of 2000 cells per well in 384 well plates using 30 mul total volume (cells plus media) per well. This will allow us to use a volume of 100 nl for pinning of the compounds 24 hours later. The cells will be incubated with test molecules for 24 hours and the luciferase activity will be detected using 20 mul per well of the Steady Glo Reagent from Perkin Elmer. The positive control is 27 uM parthenolide which is known to decrease TRPM-1 expression and kill melanoma cells.Expected outcome:Compounds that inhibit MITF directly or indirectly will reduce the expression of TRPM-1 expression and lead to a reduction in luminescence signal. Compounds that increase the activity of MITF or somehow lead to increased expression of TRPM-1 will lead to an increase in luminescence signal.MITF Primary Screening Protocol(TRPM-1 Promoter//Luciferase reporter assay)Day 1, plate TRPM-1 luc/SKMEL5 cells at 2000 per well in 30 uL media (phenol red free DMEM/10% heat inactivated Fetal Bovine Serum/Penicillin/Streptomycin/L-Glutamine)Day 2, pin 100 nL compound/DMSO solution into 30 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates. (For HTS, will require sentinel pinning with the positive control, parthenolide)Incubate 24 hours at 37 degrees C in Liconic incubator.Day 3, add 20 uL 100% Promega SteadyGlo per well with Thermo Combi fluid transfer apparatus.Shake 15 seconds on "big bear" plate shaker and incubate at room temperature for 5 minutes.Read on the Perkin-Elmer EnVision plate reader with ultra-sensitive luminescence (US LUM) settings for 0.5 sec per wellPRESENCE OF CONTROLS: Neutral control wells (NC; n=104) and positive control wells (PC; n=20) were included on every plate.EXPECTED OUTCOME: Active compounds result in decreasing readout signal.The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:ACTIVE CONCENTRATION LIMIT:For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).NORMALIZATION:The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.Experimental wells values were scaled to this range.PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).AC values were calculated up to the active concentration limit described for each sample.pAC was set to equal -1*log10(AC)PUBCHEM_ACTIVITY_OUTCOME:Activity_Outcome = 1 (inactive) when:a) compound shows activity but in a direction opposite to the expected outcomein these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to nullb) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, orc) AC > AC_limitActivity_Outcome = 2 (active) when:AC <= AC_limitActivity_Outcome = 3 (inconclusive) when:a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, orb) The fit was deemed not valid due to poor fit quality.PUBCHEM_ACTIVITY_SCORE:If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),then PUBCHEM_ACTIVITY_SCORE = 0If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)Scores relate to AC in this manner:120 = 1 pM90 = 1 nM60 = 1 uM30 = 1 mM0 = 1 MWhen the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.DATA AGGREGATION:Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:a. '2' (active) when all test outcomes were '2' (active), orb. '1' (inactive) when all test outcomes were '1' (inactive), orc. '3' (inconclusive) when the test outcomes were mixed.2. The final ACTIVE_CONCENTRATION (AC) was set as follows:a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.Note:The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.488944Summary assay4286human MITF40807040MITF microphthalmia-associated transcription factor [ Homo sapiens ]16899407Review of MITF function16001072Implication of MITF in melanomahttp://www.broadinstitute.orgBroad Research Institute1AC50_Qualifier'>','=', or '<'42542AC50_uMThe concentration at which activity reaches 50% of the maximum153pAC50_MEqual to -1*log10(AC50)42544ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.22545ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.22546ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.42547AC50_Qualifier_TEST1'>','=', or '<'42548AC50_uM_TEST1The concentration at which activity reaches 50% of the maximum159pAC50_M_TEST1Equal to -1*log10(AC50)425410Hill_Slope_TEST1The slope at AC50125411S0_(%)_TEST1The fitted activity value at zero concentration11512Sinf_(%)_TEST1The fitted activity value at infinite concentration11513Num_Points_TEST1The number of data points used to generate the plot225414Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentration11515Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observed1516Max_Concentration_uM_TEST1Maximum valid test concentration1517Activity_at_0.000060uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1155.99999984842725e-055118Activity_at_0.00012uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.0001199999969685455119Activity_at_0.00026uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.0002600000007078055120Activity_at_0.0005uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.0005000000237487265121Activity_at_0.001uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.001000000047497455122Activity_at_0.0021uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.002099999925121675123Activity_at_0.0042uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.004199999850243335124Activity_at_0.008uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.008000000379979615125Activity_at_0.016uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.01600000075995925126Activity_at_0.03uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.02999999932944775127Activity_at_0.068uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.0680000036954885128Activity_at_0.135uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.1350000053644185129Activity_at_0.26uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.2599999904632575130Activity_at_0.5uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1150.55131Activity_at_1uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration11515132Activity_at_2.1uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1152.099999904632575133Activity_at_4.2uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration1154.199999809265145134Activity_at_8uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration11585135Activity_at_16uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration115165136Activity_at_19.5uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration11519.55137Activity_at_23.5uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration11523.55138Activity_at_26uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration115265139Activity_at_35uM_(%)_TEST1The average measured activity of all accepted replicates at the specified concentration115355140ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.225441ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.225442ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.425443AC50_Qualifier_TEST2'>','=', or '<'425444AC50_uM_TEST2The concentration at which activity reaches 50% of the maximum1545pAC50_M_TEST2Equal to -1*log10(AC50)425446Hill_Slope_TEST2The slope at AC50125447S0_(%)_TEST2The fitted activity value at zero concentration11548Sinf_(%)_TEST2The fitted activity value at infinite concentration11549Num_Points_TEST2The number of data points used to generate the plot225450Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentration11551Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observed1552Max_Concentration_uM_TEST2Maximum valid test concentration1553Activity_at_0.000060uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1155.99999984842725e-055254Activity_at_0.00012uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.0001199999969685455255Activity_at_0.00026uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.0002600000007078055256Activity_at_0.0005uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.0005000000237487265257Activity_at_0.001uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.001000000047497455258Activity_at_0.0021uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.002099999925121675259Activity_at_0.0042uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.004199999850243335260Activity_at_0.008uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.008000000379979615261Activity_at_0.016uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.01600000075995925262Activity_at_0.03uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.02999999932944775263Activity_at_0.068uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.0680000036954885264Activity_at_0.135uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.1350000053644185265Activity_at_0.26uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.2599999904632575266Activity_at_0.5uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1150.55267Activity_at_1uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration11515268Activity_at_2.1uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1152.099999904632575269Activity_at_4.2uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1154.199999809265145270Activity_at_8uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration11585271Activity_at_16uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration115165272Activity_at_19.5uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration11519.55273Activity_at_23.5uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration11523.55274Activity_at_26uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration115265275Activity_at_35uM_(%)_TEST2The average measured activity of all accepted replicates at the specified concentration1153552121CR Plot Labels 1ConcentrationResponse02CR Plot Labels 2ConcentrationResponse01 R03 DA031089-012